Mass spectrometry is widely used in analytical chemistry and other fields for identifying unknown compounds, screening for the presence of certain target compounds, identifying the products of chemical reactions, studying the kinetics or mechanisms of chemical reactions, etc. Since mass spectrometers are capable of directly detecting only ions, provision must be made for ionizing the molecular constituents of samples to be analyzed. Many different types of ion sources are available for this purpose. Various of these ion sources include thermionic cathode filaments for initiating the ionization process. An electric current (either AC or DC) is passed through the filament, which typically comprises a straight, bent or coiled wire or metal foil or ribbon. Through internal electrical resistance, the filament temperature is caused to increase and, in operation, at least a portion of the filament will become sufficiently hot such that electrons are emitted from that portion. If the filament is configured as the cathode member of a cathode/anode pair, the emitted electrons can be accelerated and directed as a beam into an ionization volume within which they may react with various gaseous molecules so as to form ionized chemical species. The electrons emitted from the filament (emission current range 5-500 μA, most commonly 50 μA) are accelerated to kinetic energies of 20 to 150 eV (typically 70 eV) which is generally optimal for ionizing and fragmenting molecules.
Thermionic cathode filaments, as described above, are used in electron ionization (EI) ion sources (referred to as “electron impact” ion sources in some literature), in chemical ionization (CI) ion sources, in ion sources used to produce reagent ions for electron transfer dissociation (ETD) ion-ion reactions and in ion sources used to produce reagent ions for proton transfer reaction (PTR) ion-ion reactions. In the course of operation, various gases are introduced into or are present in such ion sources. These include various “background” gases which, according to the terminology used in this document, are gases that are introduced or are present at moderately high pressures (0.1-2.0 Torr). Such background gases thus include methane (CH4), nitrogen (N2) isobutane (C4H10) and ammonia (NH3). It should be noted that this terminology is slightly different from that usually employed in chemical ionization literature, in which methane, isobutane and ammonia are usually referred to as “reagent” gases. As used in this document, the term “reagent”, as applied to gases, is restricted to those gases that are employed as precursors for formation of reagent ions that are employed in ion sources used to produce reagent ions for ETD or PTR ion-ion reactions. Such reagent gases are introduced at low pressures—less than approximately 1×10−5 Torr. Additionally, sample gases are introduced into EI and CI ion sources. Sample gases generally include analyte compounds as well as matrix compounds.
In electron ionization (EI) ion sources, the electrons emitted from the thermionic filament are caused to directly impinge upon gaseous molecules of one or more chemical constituents of a sample under investigation. These chemical constituents may include one or more analyte compounds or matrix compounds derived from the sample. The interaction of energetic electrons of the electron beam with an electron cloud of a neutral molecule may effectively “dislodge” one or more electrons of the neutral molecule and may additionally cleave chemical bonds. This combination of ionization and fragmentation may lead to the formation of one or more cation species. It is these cation species that are analyzed by a mass analyzer. Such electron ionization sources are commonly employed in gas chromatography/mass spectrometry (GCMS) instruments.
In chemical ionization (CI) ion sources, a beam of accelerated electrons emitted from a thermionic filament is caused to interact with a low-molecular-weight (less than approximately 50 Da) background gas such as methane, nitrogen, iso-butane or ammonia supplied in relatively high abundance in comparison to the sample molecules. The interaction between such background gas and the introduced electrons may include a complex set of primary and secondary electron-molecule and ion-molecule reactions so as to form a variety of molecular and ionic species. These reactions may also result in a population of near-thermal-energy electrons. The ions formed from the background gas may include species such as CH3+, CH4+•, CH5+ and C2H5+ (in the case of methane background gas); iso-C4H10+• and iso-C4H9+ (in the case of iso-butane background gas); and NH3+•, NH4+ and N2H7+ (in the case of ammonia background gas). Analyte ions—generally cations—may be created by reaction between analyte molecules and one or more of the ionic species derived from the background gas. In the case of electronegative analyte molecules, electron capture of near-thermal-energy electrons produced upon electron ionization of background gas by the accelerated electrons can yield analyte anions.
In some types of applications, a CI-type source may be employed so as to produce reagent ions for the purpose of conducting ion-ion reactions within a separate ion-ion reaction cell or region of a mass spectrometer apparatus. The reagent ions are derived from a reagent precursor material that is introduced in gas form into an ionization volume of the CI-type source. After generation, the reagent ions are subsequently transferred to and employed in a reaction cell where they are caused to react with sample-derived precursor ions so as to induce dissociation or charge reduction of the precursor ions. The diagnostic ions are thus, in this case, product ions that are formed by reaction between the reagent ions and the sample-derived precursor ions in the ion-ion reaction cell, chamber or region. In such applications, the sample constituents are initially ionized using another separate ion source of the mass spectrometer apparatus. The mass spectrometer apparatus will thus include two ion sources—a first ion source (typically an electrospray ionization source) for initially ionizing the sample-derived materials and a second CI-type source, here referred to as a reagent ion source, for generating reagent ions.
Electron transfer dissociation (ETD) is a first type of application that employs a reagent ion source—in this case, an ETD source. The ETD technique is described by Hunt et al. in U.S. Pat. No. 7,534,622 for “Electron Transfer Dissociation for Biopolymer Sequence Mass Spectrometric Analysis”, by Syka et al. in “Peptide and Protein Sequence Analysis by Electron Transfer Dissociation Mass Spectrometry”, Proc. Nat. Acad. Sci., vol. 101, no. 26, pp. 9528-9533 (2004), by Coon et al. in “Anion Dependence in the Partitioning Between Proton and Electron Transfer in Ion/Ion Reactions”, Int. J. Mass Spectrometry, vol. 236, nos. 1-3, pp. 33-42 (2004), by Syka et al. in International Application Publication WO 2011/028863 A1, by Hartmer in U.S. Patent Application Publication No. 20100140466 A1 and by Brown et al. in International Application Publication WO 2011/092515 A1, all of which are incorporated herein by reference. Proton transfer reaction (PTR) is a second type of application that employs a reagent ion source (in this case, a PTR source). The PTR technique is described by Hunt et al. in U.S. Pat. No. 7,749,769 for “Simultaneous Sequence Analysis of Amino- and Carboxy-Termini” as well as in Stephenson and McLuckey, Anal. Chem. 1996, 68, pp. 4026-4032 and McLuckey and Stephenson, Mass Spectrometry Reviews 1998, 17, pp. 369-407.
In either the ETD or the PTR technique, near-thermal-kinetic-energy electrons produced during electron ionization of a background gas such as nitrogen are captured, in an ETD or PTR ion source, by molecules of a reagent gas so as to produce reagent anions. The reagent anions are then transferred out of the ion source and into a reaction cell in which they are allowed to react with ions of an analyte.
In the ETD technique, the anions are reacted with multiply charged analyte cations (for example, multiply protonated peptide/protein molecules) so as to transfer an electron from the reagent anion to the analyte cation, thereby inducing dissociation of the analyte cation. As discussed in U.S. Pat. No. 7,534,622, many gaseous polycyclic aromatic hydrocarbon species (also known as “polyaromatic hydrocarbon” species) may be suitably employed as ETD reagents (reagent-ion precursors). These include but are not necessarily limited to the compounds naphthalene; fluorine; phenanthrene; pyrene; fluoranthene; chrysene; triphenylene; perylene; acridine; 2,2′ dipyridyl; 2,2′ biquinoline; 9-anthracenecarbonitrile; dibenzothiophene; 1,10′-phenanthroline; 9′ anthracenecarbonitrile; anthraquinone and substituted derivatives of these compounds.
Recently, Syka et al. (International Application Publication WO 2011/028863 A1) have taught the use of certain additional polycyclic aromatic hydrocarbon species as ETD reagents. These new ETD reagent compounds include azulene, homoazulene, acenaphthylene, homodimers of any of azulene, homoazulene, or acenaphthylene and heterodimers comprising one each of azulene, homoazulene, or acenaphthylene. Additionally, Brown et al. (International Application Publication WO 2011/092515 A1) have recently taught the use, as ETD reagents, of certain unsaturated organic compounds having respective Franck-Condon factors between 0.1 and 1.0 and positive electron affinity (EA) values of between 0.1 to 200 kJ/mol. As is known, the Franck-Condon factor is a measure of the overlap of the vibrational wavefunctions of neutral and anionic species. The ETD reagents taught by Brown et al. include the aromatic (not polycyclic aromatic) compounds nitrosobenzene; nitrobenzene; 1,4 dicyanobenzene; 1,3 dicyanobenzene; 5-cyano 1,2,4-triazole; amitrole 2-aminopyridine; 2-pyridine carbonitrile; 3-pyridine carbonitrile; 3-chlorobenzonitrile; 4-chlorobenzonitrile; 3-pyridinecarbonitrile; and 4-pyridinecarbonitrile. The ETD reagents taught by Brown et al. further include the non-aromatic compounds 1,2-dicyanoethylene and 1,2-dicyanoacetylene.
Still further, Hartmer (U.S. Patent Application Publication No. 20100140466 A1) has recently taught that certain aliphatic compounds having electron affinity values between 0.3 and 0.8 electron-volts may also be employed as ETD reagents (reagent-ion precursor gases). Hartmer notes that certain aliphatic compounds having double bonds, particularly polyenes, are especially favorable in this regard. Hartmer further notes that the compound un-substituted 1,3,5,7-cyclooctatetraene (C8H8) is an aliphatic polyene that is a particularly favorable compound for use as a reagent-ion precursor gas for ETD. Hartmer further notes that some forms of 1,3,5,7-cyclooctatetraene obtained through alkyl substitution (alkyl=methyl, ethyl, propyl, isobutyl), and some forms of cyclooctatetraene with heteroatoms are also suitable for the creation of ETD reagent ions.
In the PTR technique, multiply charged analyte cations (for example, peptide/protein cations) are reacted with an anion that removes protons from the analyte cation, thereby reducing the net charge of the analyte cation without causing fragmentation. In this fashion, the charge state of the ion can be determined. Benzoic acid (C6H5COOH) is commonly used as a reagent for PTR and has been observed to promote growth of carbonaceous masses on thermionic filaments. As discussed in U.S. Pat. No. 7,749,769, other suitable PTR reagents include, but are not limited to, perfluoro-1,3-dimethylcyclohexane, sulfur hexafluoride, and perfluorotributylamine.
In general, relatively modest flows (typically on the order of 0.2 to 5 atm-mL/minute) of low molecular weight background gases (approximately less than 50 Da), such as those listed above, can pressurize the ionization region of a CI-type source to about 0.1 to 2 Torr. The electron stream emanating from the filament is directed into the interior of an ionization volume into which the high abundance background gas is admitted. If the CI-type source is used to ionize sample-derived compounds, molecules of the sample material are also admitted into the ionization volume in the gas phase. In this case, ions formed from the background gas react with the sample or analyte molecules so as to yield sample ions.
In ETD or PTR reagent ion sources, molecules of the reagent precursor gas (but not sample-derived compounds) are introduced into the ionization volume in the gas phase. The reagent precursor gas may typically comprise an aromatic or poly-aromatic compound but is not limited to these classes of chemical compounds. In the ionization volume, near-thermal electrons are captured by the reagent molecules (i.e., via electron capture ionization) to produce reagent anions, which are then conveyed to the appropriate reaction cell of a mass spectrometer for reaction with analyte ions. In this ETD-type or PTR-type of application, typical background or reagent gas (typically nitrogen or methane) pressures for achieving maximum reagent ion generation are typically in the range of 0.1 to 0.4 Torr.
Each of the ion-source types described above typically employs a thermionic filament to generate the electron beam that initiates ionization. Because a gas diffusion or gas flow pathway will generally exist between the hot, electron-emitting portion of the filament and the gas or gases that are to be ionized, it is not possible to fully shield this filament portion from coming into contact with possible contaminating molecules or ions. Exposure to some of these gases can have a deleterious effect on the structural integrity and lifetime of a filament. Such filament-compromising gas species may be derived from a sample component such as an analyte or matrix compound (in the case of EI sources or CI sources used to ionize analytes), from a reagent gas (in the case of ETD or PTR reagent ion sources) or a carbon-containing background gas. The element carbon is the most abundant element of many of such sample or reagent materials. Unfortunately, the solubility of carbon in common filament materials is non-negligible in the temperature range (at or above approximately 1200 K, but typically 2200-2700 K) necessary to achieve suitable electron beam currents for the above-described ion sources.
In accordance with the above considerations, it has been observed that when a filament-based ion source is employed in the various manners described above to generate ions, its filament may exhibit the formation of a carbonaceous growth adjacent to one or both terminating poles, resulting in the eventual failure of the filament. Such carbonaceous masses are herein referred to as “tumors”. Without reliance on any particular theory, it is believed that carbonaceous growth formation arises from the diffusion and/or electromigration of dissolved carbon in the filament. As the carbon reaches the cooler portion of the negative leg (using a direct-current source) or either leg (using an alternating-current source) of the filament, it is believed that its electromobility through and solubility within the metal filament drops and, consequently, the carbon precipitates from solution and accumulates. The precipitation of the carbon-bearing phase or phases disrupts the structural integrity of the metal filament, ultimately causing its failure.
The primary source of the dissolved carbon has been experimentally demonstrated to be the sample, sample matrix or reagent molecules that diffuse to and react with the filament. The applicants believe that these carbon-containing molecules decompose upon contact with the filament when the filament is heated to the temperatures necessary to promote electron emission and the resultant carbon atoms diffuse into the bulk filament material. It has been found that such carbonaceous tumor growth can occur on filaments within electron ionization (EI) and CI ion sources, which are most commonly employed in gas chromatography mass spectrometry (GC-MS) applications. In such cases, a carbon-containing sample material, analyte and/or matrix, is believed to be the source of the carbon that causes the tumor growth.
The growth of carbonaceous tumors on ion-source filaments is believed by the inventors to be a general phenomenon that occurs when such filaments are exposed to almost any carbon-containing gas or gases. The inventors have observed such carbonaceous tumor growth on filaments exposed to the ETD reagent gases fluoranthene and phenanthrene, to the PTR reagent gas benzoic acid, to the common calibration gas perfluorotributylamine, as well as to, in EI applications, matrix compounds in gases derived from various silicone samples and food samples. Although carbon containing background or reagent gases have the potential to deliver carbon to filaments, in the inventors' experience, methane, the most widely used background gas for CI applications, does not induce the growth of carbonaceous tumors on filaments. However, many other background or reagent gases are likely to do so.
The above-described “tumor growth” failure mode is distinguishable from the “normal” filament failure mode, caused by the gradual sublimation of the filament material (e.g., rhenium) at hottest point on the filament where the majority of the electron emission occurs (e.g., proximate to the tip or apex of the filament for hair pin type filaments). The occurrence of carbonaceous growth formation has a strong likelihood of significantly shortening the filament lifetime, thereby increasing instrument downtime due to the need to replace the filament at relatively frequent intervals. The problem of carbonaceous growth formation and consequent filament failure is exacerbated by the conditions at which the CI-type source is typically operated for generating reagent ions (such as PTR and ETD reagent ions) for ion-ion reaction applications. More specifically, the source is operated as a “bright” (relatively high ion production rate, usually in excess of 3×107 reagent ions/second) reagent ion source to minimize the time required to generate the requisite numbers of reagent ions, thereby enabling relatively high numbers of ETD MS/MS experiments to be conducted in a given time. Furthermore, in the context of large-scale proteomics studies, the source may be operated on a near-continuous basis over multiple days. The foregoing and other factors may tend to promote and accelerate the processes that produce carbonaceous growth formation.